Vaccine composition may comprise one or more of adjuvants. ‘Adjuvants’ are substances that are incorporated into, or injected simultaneously, with an antigen and that potentiate non-specifically the ensuing immune responses. The resultant immune responses last longer by maintenance of sufficient levels of antibodies in the administered population. For practical and economic reasons, this prophylactic immunization needs to be obtained with minimum number of administrations and employing least amount of antigen compatible with efficient immunization. The nature of these adjuvants may be inorganic like alum, such as aluminium phosphate and aluminium hydroxide which are most commonly used in human vaccines, and organic adjuvants like squalene.
Veterinary vaccines commonly make use of oil-based adjuvants. A vaccine used for prevention of influenza caused by H5N1 virus, which is commonly referred to as an avian influenza or “bird flu”, contains the adjuvant AS03, an oil-in-water emulsion. The AS03 adjuvant is made up of the oily compounds D, L-alpha-tocopherol (vitamin E), squalene, an emulsifier—polysorbate-80, which helps ingredients to mix together and keep them from separating, and water containing small amounts of salts.
Aluminium salts are widely used, since 1930s. The only adjuvants approved by the Food and Drug Administration for use in human vaccines are aluminium-containing adjuvants, due their long history of safe and effective use. Glenny et al., had described the effect of aluminium compounds as adjuvant (Glenny A T, Pope C G Waddington H, Wallace U. Immunological Notes XVII to XXIV. J. Pathol. 29, 31-40, 1926).
Despite this, aluminium-containing adjuvants have been described as being difficult to manufacture with reproducible physicochemical properties. Scholtz et al., in 1984, prepared pure aluminium phosphate using equimolar amounts of aluminium chloride and trisodium phosphate.
Aluminium Phosphate gel is used as the ‘adjuvant’ in the formulations of the liquid pentavalent vaccine (LPV), helping to boost the immunogenic responses to Hepatitis-B Surface Antigen, Diphtheria and Tetanus toxoids which get adsorbed onto the gel particles, and also possibly for the whole-cell Pertussis antigens. The aluminium salts are used in. DTaP vaccines, the pneumococcal conjugate vaccine and hepatitis-B vaccines. Although there has been a search for alternate adjuvants, aluminium compounds (aluminium phosphate and hydroxide) will continue to be used as adjuvants for human vaccines for many years owing to their good track record of safety, low cost and adjuvanticity with a variety of antigens.
Two methods have been commonly used to prepare vaccines and toxoids with aluminium compounds—in situ precipitation of aluminium compounds in the presence of antigen (developed originally to purify toxoids by precipitation with alum), and adsorption of antigen onto preformed aluminium gel. Adsorption of antigens on aluminium adjuvants, either during in situ precipitation of aluminium adjuvants or onto preformed aluminium gels, depends upon physical and chemical characteristics of antigen, type of aluminium adjuvant and conditions of adsorption. These conditions are often overlooked, and a poorly formulated aluminium adjuvant preparation does not exhibit optimal adjuvanticity.
SU481539 disclosed a method of producing a porous hydrogel alumino-phosphate by reacting aluminium chloride with 85% phosphoric acid. The resulting solution was cooled to −8 to −10° C., slowly introduced with vigorous stirring, cooled to the same temperature as ethylene oxide. The resulting gel was heated at a temperature of 50-350° C. and a water vapour pressure of 1-170 atm for 2 hours, washed with distilled water, dried and calcined at 200° C. in air at 650-700° C. for 4-6 hours.
SU550340 disclosed a method of producing aluminium phosphate gel by reacting aluminium acetate with phosphoric acid, followed by filtration, washing, drying at 30-40° C. for 12 hours, then at 110-120° C. for 4 hours and at 600° C. product activation within 4 hours. The proposed method is also complicated and laborious, because it requires prolonged drying time and the activation of the final product at high temperatures.
SU559895 disclosed a method of producing an amorphous aluminium phosphate hydrate by reacting a solution of aluminium nitrate and phosphoric acid in the molar ratio 1:0.95-1.05, followed by neutralization with ammonia to pH=6.0 with temperature in the range of 15-20° C. The resulting product is filtered, washed with water and dried at a temperature of 60-80° C.
DE 2152228 disclosed a process, for the production of aluminium phosphate gel, which comprises of forming a mixture of sodium aluminate, phosphoric acid and aluminium sulphate in aqueous medium, reacting the mixture in such a way that the pH value of the resulting suspension is between 5 and 6, and heating the aluminium phosphate, which is precipitated, to a temperature above 70° C. either during or after its precipitation.
RU2149138C disclosed a process of producing Aluminium phosphate gel wherein an initial solution of water-soluble salts of aluminium and sodium phosphate was subjected to filtration on microfiltration unit with a threshold bandwidth of 0.22 microns, and reacted with a soluble aluminium salt with sodium phosphate. The desired product was precipitated under vigorous stirring for 15-45 min. at a rotation speed of the stirrer of 3.3-8.3 sec−1. Aluminium phosphate gel is formed at a temperature of 18-60° C. for 5-7 days, followed by its washing.
WO 2009/136233A1 disclosed method for production of nanoparticles of aluminium phosphate with particle diameter less than 1000 nm, preferably 10 to 600 nm, comprising: a. preparation of aluminium phosphate gel; b. adjusting pH of the aluminium phosphate gel; c. subjecting the aluminium phosphate gel to size reduction; d. affording aluminium phosphate nanoparticles of desired size; and e. optionally suspending the nanoparticles in a suitable buffer, wherein the aluminium phosphate gel can be prepared (i) in situ, (ii) by suspending aluminium phosphate powder in suitable solvent, or (iii) by treatment of equimolar aluminium chloride with trisodium phosphate to effect aluminium phosphate gel formation, followed by chloride removal, if required.
U.S. Pat. No. 8,540,955 disclosed an improved method, for producing the aluminium adjuvant AlPO4, which comprises the steps of mixing a solution of aluminium chloride and a solution of sodium phosphate tribasic to produce an aluminium phosphate precipitate, wherein the improvement comprises settling the aluminium phosphate precipitate at a temperature in the range of about 50° C. to about 70° C.
Burrell et al. [Vaccine. 1999 Jun. 4;17(20-21):2599-603] disclosed that Aluminium phosphate adjuvant remained amorphous when autoclaved for 30 or 60 min. at 121° C. However, deprotonation and dehydration reactions occurred as evidenced by a decrease in the pH. The protein adsorption capacity, rate of acid neutralization at pH 2.5 and point-of-zero charge also decreased, indicating that the deprotonation/dehydration reactions resulted in a decreased surface area.
Buttell et al. [Vaccine. 2000 Sep. 15;19(2-3):275-81] disclosed a process for preparing Aluminium phosphate adjuvant wherein an aqueous solution containing aluminium chloride and sodium dihydrogen phosphate was pumped into the reaction vessel at a constant rate. A second pump infused a sodium hydroxide solution at the rate required to maintain the desired pH. Precipitations were performed between pH 3.0 and 7.5, at intervals of pH 0.
The characteristics of aluminium adjuvants, such as size of the gel particles, adsorption capacity, isoelectric point, and ratio of aluminium to phosphate depend upon the conditions of making these gels, including order of adding reagents, speed at which the reagents are added & mixed, mixing speed, time taken to adjust pH, and scale of gel preparation. Therefore, aluminium adjuvants have been described as difficult to manufacture in a physico-chemically reproducible way, thus resulting in batch to batch variations.
The methods of preparing aluminium phosphate gel disclosed in the above prior arts are tedious and complex. Although methods for producing aluminium phosphate adjuvant have been described, there remains a need in the art for methods that are more efficient on an industrial scale. In addition, it is desirable that the characteristics of aluminium phosphate adjuvant produced by any new method should satisfy the properties of the adjuvant already present in various marketed products to enable its usage in vaccine preparations.